SIGN-R1 contributes to protection against lethal pneumococcal infection in mice

Abstract

Rapid clearance of pathogens is essential for successful control of pyogenic bacterial infection. Previous experiments have shown that antibody to specific intracellular adhesion molecule-grabbing nonintegrin (SIGN)-R1 inhibits uptake of capsular polysaccharide by marginal zone macrophages, suggesting a role for SIGN-R1 in this process. We now demonstrate that mice lacking SIGN-R1 (a mouse homologue of human dendritic cell-SIGN receptor) are significantly more susceptible to Streptococcus pneumoniae infection and fail to clear S. pneumoniae from the circulation. Marginal zone and peritoneal macrophages show impaired bacterial recognition associated with an inability to bind T-independent type 2 antigens such as dextran. Our work represents the first evidence for a protective in vivo role for a SIGN family molecule.

Figure 1.

Inactivation of the mouse SIGN-R1 gene by homologous recombination. (a) Structure of the human DC-SIGN locus. (b) Structure of the mouse SIGN-R1 locus, the targeting vector, and the predicted homologous recombination event are shown. NeoR, neomycin resistance cassette; TK, thymidine kinase cassette; B, BamHI. (c) RT-PCR analysis of SIGN-R1 expression. RNA was prepared from splenocytes and PCR was performed. ^−/−, SIGN-R1^−/−. (d) RT-PCR analysis of SIGN-R1 expression using PCR primers throughout the cDNA to verify gene deletion. (1) Wild-type. (2) SIGN-R1^−/−. (3) Wild-type. (4) SIGN-R1^−/−. (e) Staining with ER-TR9 (anti–SIGN-R1) monoclonal antibody using horseradish peroxidase detection. (f) Co-staining with ER-TR9 monoclonal antibody (Texas red) and MOMA monoclonal antibody (FITC) (identifying marginal metallophilic macrophages). Original magnification, 10.

Figure 2.

Analysis of MZMs and ALEXA-dextran uptake in the spleens of wild-type (WT) and SIGN-R1^−/− mice. (a and b) ALEXA-dextran (Dex, green) localized to the MZs in spleen sections 2 h after i.v. inoculation. (c and d) Spleen sections demonstrating ALEXA-dextran uptake counterstained with anti-CD11b (Texas red). (e and f) Spleen sections demonstrating ALEXA-dextran uptake counterstained with ER-TR9 antibody (anti–SIGN-R1; Texas red). (g and h) Spleen sections showing results of coinoculation with ALEXA-dextran (green) and Indian ink (black spots). (i and j) Spleen sections demonstrating anti-MARCO antibody (FITC) staining of MZM. All original magnifications,10.

Figure 3.

Absence of SIGN-R1 increases susceptibility to pneumococcal peritonitis. (a) Survival after S. pneumoniae type 2 infection. SIGN-R1^−/− mice (n = 12) or control mice (n = 12) were inoculated intraperitoneally with 10³ S. pneumoniae type 2. SIGN-R1^−/− have reduced survival (P = 0.005). The experiment shown is representative of two, and p-values were obtained using a Log rank test. (b and c) Physical signs of illness in mice with pneumococcal peritonitis. After inoculation with S. pneumoniae (type 2), mice were monitored by a blinded observer for the signs of systemic illness. The presence or absence of piloerection (1 point), with additional hunched posture (2 points), and lack of spontaneous movement (3 points) was scored in control and SIGN-R1^−/− mice inoculated with 10³ S. pneumoniae at 24 h (b) and 30 h (c). The experiment is representative of two. (d) 30 h after inoculation with S. pneumoniae, tail bleeds were performed on control (n = 12) and SIGN-R1^−/− mice (n = 10), and blood was cultured for bacterial growth. More SIGN-R1^−/− mice were bacteremic (P = 0.014); the p-value was obtained using a Fisher's exact test. (e) Survival after S. pneumoniae type 14 infection. SIGN-R1^−/− mice (n = 10) or control mice (n = 10) were inoculated intraperioneally with 10⁵ S. pneumoniae type 14. SIGN-R1^−/− have reduced survival (P = 0.018). Numbers denote individual mice. p-values were obtained using a Log rank test. (f and g) Physical signs of illness in mice with pneumococcal peritonitis. After inoculation with S. pneumoniae (type 14), mice were monitored by a blinded observer for the signs of systemic illness (as described before). (h) 26 h after inoculation with S. pneumoniae, tail bleeds were performed on control (n = 10) and SIGN-R1^−/− mice (n = 10), and blood was cultured for bacterial growth. More SIGN-R1^−/− mice were bacteremic (P = 0.023).

Figure 4.

Absence of SIGN-R1 does not impair natural antibody levels or opsonization of S. pneumoniae. (a) Natural anti-PC antibody levels in control and SIGN-R1^−/− mice. Sera from naive WT and SIGN-R1^−/− mice were assayed for natural antibodies against PC by ELISA. Each point represents the value from an individual mouse. p-values were determined using an unpaired Student's t test (Welch corrected). (b and c) Nonimmune serum from control and SIGN-R1^−/− mice provide equivalent opsonization. The RAW-297 macrophage cell line (b) or BALB/c peritoneal macrophages (c) were incubated at 37°C with FITC-labeled S. pneumoniae opsonized with heat-inactivated nonimmune serum (NIS) from wild-type or SIGN-R1^−/− mice, followed by flow cytometric analysis. Phagocytosis is expressed as percentage of FITC positive cells. Values represent mean of triplicates, the experiments shown are representative of two, and p-values were obtained using an unpaired Student's t test.

Figure 5.

Impaired recognition of S. pneumoniae by SIGN-R1^−/− peritoneal macrophages. (a) S. pneumoniae bind preferentially to SIGN-R1–transduced NIH3T3 (SIGNR1) cells compared with wild-type (WT) NIH3T3. Cells were incubated for 1 h at 4°C with FITC-labeled pneumococci, washed, and analyzed by flow cytometry. Values represent the mean of triplicates, and p-values were obtained using an unpaired Student's t test. (b–d) Pooled peritoneal macrophages from wild-type (n = 2) or SIGN-R1^−/− mice (n = 2) were incubated for 30 min with FITC-labeled S. pneumoniae at 4°C to assess binding (b), or 37°C to assess phagocytosis (c), followed by flow cytometric analysis. Binding or phagocytosis are expressed as percentage of FITC positive cells. SIGN-R1^−/− peritoneal macrophages show reduced binding and phagocytosis of S. pneumoniae. Representative FACS profiles are shown. Values represent mean of triplicates, the experiments shown are representative of two, and p-values were obtained using an unpaired Student's t test. (d) Confocal images of wild-type and SIGN-R1^−/− peritoneal macrophages labeled with Mac-1 allophycocyanin after incubation with FITC-labeled S. pneumoniae at 37°C for 45 min.

Figure 6.

Impaired S. pneumoniae trapping by SIGN-R1^−/− MZM. Wild-type (left) and SIGN-R1^−/− (right) mice were injected intravenously with 10⁷ FITC-labeled S. pneumoniae. 30 min later, spleens were snap frozen, and cryosections were prepared and counterstained with anti-MARCO antibody (Texas red) to delineate MZMs. Arrows indicate areas where S. pneumoniae are located in the red pulp and are not restricted to the MZ. FO, follicles. Original magnification, 10 (top), 20 (bottom). Representative sections from two mice per group are shown.